非瑟酮通过Nrf2/HO-1和MAPK信号通路抑制阿霉素诱导的肺动脉内皮细胞衰老  

Fisetin Inhibits Doxorubicin-induced Senescence of Pulmonary Artery Endothelial Cells Through Nrf2/HO-1 and MAPK Signaling Pathways

作　　者：高雨彤 肖娜[2] 敖启林[3] Gao Yutong;Xiao Na;Ao Qilin(Department of Pathology Union Hospital,Tongji Medical College,Huazhong University of Science and Technology Wuhan 430022,China;Department of Pathology,The First People S Hospital of Xiantao Yangtze University,Xiantao 433000,China;Department of Pathology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属协和医院病理科,武汉430022 [2]长江大学附属仙桃市第一人民医院病理科,仙桃433000 [3]华中科技大学同济医学院附属同济医院病理科,武汉430030

出　　处：《华中科技大学学报（医学版）》2021年第6期685-692,共8页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81270105)。

摘　　要：目的探讨非瑟酮(Fisetin)对阿霉素(Doxorubicin,DOX)诱导的人肺动脉内皮细胞(HPAEC)衰老的影响及其相关机制。方法使用不同浓度的DOX(0.1、0.25、0.5、1μmol/L)诱导HPAEC衰老,通过CCK8法检测细胞增殖活性,Westernblot法检测衰老相关蛋白P53、P21表达。随后用不同浓度的Fisetin作用于HPAEC,分别于0、24、48和72 h检测Fisetin对HPAEC细胞增殖的影响,流式细胞术检测Fisetin对细胞周期的影响,利用衰老相关β-半乳糖苷酶(SA-β-gal)对衰老细胞进行染色并计数,活性氧(ROS)试剂盒检测Fisetin对HPAEC活性氧(ROS)产生的影响,Westernblot法检测衰老相关蛋白、Nrf2和MAPK信号蛋白的表达情况,酶联免疫吸附试验(ELISA)检测分泌的细胞因子浓度,CCK8法检测Fisetin预处理的HPAEC培养上清对人肺动脉平滑肌细胞(HPASMC)增殖的影响。结果当Fisetin浓度在20μmol/L以下时,HPAEC增殖活性在24、48、72h没有明显降低,差异无统计学意义(P>0.05);流式细胞术检测发现DOX刺激后,90%以上的HPAEC被阻滞在DNA合成前期(G_(0)/G_(1)期),而Fisetin(5、10、20μmol/L)处理组细胞周期在DNA合成期(S期)分别占40%、50%及55%,呈浓度依赖性上升,差异具有统计学意义(P<0.01)。Western blot证实DOX刺激后,HPAEC中衰老相关蛋白P21表达增加,而随着Fisetin作用浓度的增加,P21表达逐渐下降(P<0.05)。SA-β-gal衰老染色和ROS染色证实DOX干预后,衰老细胞的数量明显增加,ROS的产生量也增加;而Fisetin处理后,衰老细胞的数量逐渐减少,ROS的产生量也减少。Fisetin可增加HO-1、p-P38的表达,促进Nrf2核转移,沉默HO-1和抑制p-P38表达均可减弱Fisetin对P21的抑制作用。DOX刺激后细胞衰老相关分泌表型(SASP)的细胞因子表达不断升高,而不同浓度的Fisetin(5、10、20μmol/L)干预后,SASP细胞因子的表达量下降。CCK8实验证实,Fisetin预处理的HPAEC培养上清对HPASMC增殖有抑制作用。结论Fisetin可通过Nrf2/HO-1和MAPK信号通�Objective To investigate the protective effects and underlying mechanisms of Fisetin on senescence of human pulmonary artery endothelial cell(HPAEC)induced by doxorubicin(DOX).Methods The senescence of HPAEC was induced by different concentrations of DOX(0.1,0.25,0.5,1μmol/L).The cell activity was detected by CCK8 assay,and the expression of senescent protein P21 was detected by Western blotting.Then after the intervention of different concentrations of Fisetin,the effects of Fisetin on the proliferation of HPAECs were detected by CCK8 assay at 0,24,48,and 72 h.Flow cytometry was used to detect the effects of Fisetin on the cell cycle.Theβ-galactosidase(SA-β-gal)senescence staining was used to photograph and count senescent cells.Reactive oxygen species(ROS)kit was used to detect the effect of Fisetin on ROS of HPAEC cells.The expression levels of senescence-related proteins Nrf2,HO-1,MAPK signal proteins were detected by Western blotting.The secreted cytokine concentration was detected by enzyme-linked immunosorbent assay(ELISA).The cell proliferation activity of human pulmonary artery smooth muscle cell(HPASMC)after incubation with the supernatant of the medium of HPAEC treated with Fisetin was detected by CCK8 assay.Results When the Fisetin concentration was below 20μmol/L,the HPAEC proliferation activity did not decrease significantly at 0,24,48,72 h,and the difference was not statistically significant(P>0.05).The flow cytometry indicated that after DOX stimulation,more than 90%of HPAEC were blocked in the early DNA synthesis phase(G_(0)/G_(1)phase),while the cell cycle of the Fisetin treatment group was mainly in the DNA synthesis phase(S phase),and the difference was statistically significant(P<0.01).Western blotting confirmed that the expression of senescence-related protein P21 in HPAEC increased after DOX intervention,while the expression of P21 gradually decreased after Fisetin intervention(P<0.05).SA-β-gal senescence and ROS staining confirmed that after DOX intervention,the number of senescent ce

关 键 词：非瑟酮 阿霉素 肺动脉内皮细胞 肺血管重塑 衰老 

分 类 号：R965[医药卫生—药理学]